Glycolipid-enriched membrane domains are assembled into membrane patches by associating with the actin cytoskeleton
Glycolipid-enriched membrane domains are assembled into membrane patches by associating with the actin cytoskeleton
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DOI：
10.1006/excr.2001.5253
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发表时间：
2001-07-15
影响因子：
3.7
通讯作者：
Zavzavadjian, J
中科院分区：
文献类型：
--
作者：
Rodgers, W;Zavzavadjian, J
细胞的非离子去污剂裂解物含有糖脂富集膜（GEM）级分。已经提出GEM级分代表溶解性差的GEM微区或脂筏。然而，完整细胞中GEM结构域的性质仍然存在争议。为了使用共聚焦显微镜研究GEM相关蛋白的性质，使用p56（lck）的N-末端结构域（LckNT）将GFP靶向GEM结构域。表达LckNT-GFP的HeLa细胞的成像显示，它被靶向质膜中富含肌动蛋白的大斑块，其中含有高达五倍的蛋白质富集。双标记实验表明，补丁选择性地丰富与其他GEM相关的分子。此外，斑块对TX-100的提取具有抗性，并且通过提取胆固醇破坏GEM结构域也破坏了LckNT-GFP与F-肌动蛋白的共定位。类似于HeLa细胞中富含肌动蛋白的斑块，LckNT-GFP与刺激的T细胞中富含肌动蛋白的膜帽共定位。此外，破坏LckNT-GFP的GEM靶向信号也抑制其靶向膜！上限总之，这些发现扩展了以前的研究表明，协会的GEM域与肌动蛋白细胞骨架提供了一种机制，针对信号分子的膜补丁和帽。(C)北京：科学出版社.
Nonionic detergent lysates of cells contain at glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to ex traction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin, Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane! caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps. (C) 2001 Academic Press.